1. Field of the Invention
This invention relates to a method for haplotyping and breeding domesticated fowl for increased disease resistance. In one related aspect, the invention relates to a method for determining the Rfp-Y, B-F, B-L and B-G haplotypes of domesticated fowl, including chickens. In a further, related aspect, the invention relates to a method for breeding domesticated fowl raised for meat and eggs to achieve increased disease resistance and vitality.
2. Description of the Background Art
In domesticated fowl, the major histocompatibility complex (xe2x80x9cMhcxe2x80x9d) which is associated with the regulation of immune recognition and immune response, is called the B system. This system, which comprises polymorphic Mhc Class I (B-F), Mhc Class II (B-L) and B-G genes, has been known to exist since the early 1940""s. Briles, W. E. et al., Genetics, 35:633-652 (1950), Pink, J. R. L. et al., Immunogenetics, 5:203 (1977). Genotyping birds for the B system of histocompatibility has been accomplished by several different kinds of tests. The first, and by far the most commonly used method, is a serological test: hemagglutination of chicken red blood cells with alloantisera. This method requires some prior knowledge of the genetics of the animals and availability of appropriate alloantisera.
The second relies on the patterns of B-G gene restriction fragments revealed in genomic DNA digested with a restriction enzyme and analyzed in Southern hybridization with nucleic acid probes for the B-G genes. See Miller et al., U.S. Pat. No. 5,451,670. An advantage of this approach is that prior knowledge of gene sequences is not necessary. A third method relies on B-F (Class I) and B-L (Class II) gene restriction fragment patterns revealed in genomic DNA digested with several restriction enzymes and analyzed by Southern hybridization with nucleic acid probes for the B-F and B-L genes. See Lamont, S. J. et al., Poult. Sci., 69:1195 (1990). This method may underrepresent the different B-F and B-L alleles present in a population.
A fourth method is based on hybridization of oligonucleotide probes specific for known sequences in the various alleles of the B system Class I gene (gene B-FIV on the physical map of chicken Mhc genes (See Guillemot, F. et al., 1988, supra.)). This method requires knowledge of the sequence of the allele at least in the region to which the probe hybridizes. See Shuman, R. M. et al., xe2x80x9cDevelopment of an Mhc Typing Test Using DNA Amplification and Oligonuleotide Probesxe2x80x9d, Poult. Sci., 72 (Suppl. 1): 10 (Abstr.) (1993). A fifth method, also requiring knowledge of gene sequence, employs antibodies developed to a specific epitope on Class I antigens through expression of recombinant genes in chickens.
The use of a technique known as polymerase chain reaction, single-stranded conformational polymorphism (xe2x80x9cPCR-SSCPxe2x80x9d) to study the expression of B-G genes in non-erythroid tissues has been proposed. Miller and Goto, Avian Immunology in Progress, Tours (France), Aug. 31-Sep. 2, 1993, Ed. INRA, Paris 1993 (Les Colloques, No. 62). In this method, short segments of B-G genes of interest are amplified using PCR and radioactive tags. The PCR products are then denatured by heating and applied to a non-denaturing polyacrylamide gel. The single-stranded fragments of the heat-denatured DNA fragments assume secondary conformations determined by their sequences and migrate differently in the polyacrylamide gel during electrophoresis, so as to produce a pattern (or fingerprint) representative of the sequences within the genome in the region of amplification. These were revealed in films exposed to the electrophoretic gels.
Recently, a second system of major histocompatibility genes of the chicken has been discovered. Briles, W. E. et al., Immunogenetics 37:408-414 (1993). This system, designated Rfp-Y, consists of at least two Class I genes, three Class II genes and a c-type lectin gene. Miller et al., Proc. Nat""l. Acad. Sci. USA 91:4397-4401 (1994); Miller et al., Proc. Nat""l. Acad. Sci. USA 93:3958-3962 (1996). Haplotypes of Rfp-Y assort independently from haplotypes of the B system. Briles, W. E. et al., Immunogenetics 37:408-414 (1993).
The existence of a second genetically-independent complex of polymorphic histocompatibility genes was unexpected, because Mhc genes are typically considered to be in a single linkage group. Previous studies have suggested that at least one Mhc Class II gene in the B-L xcex2III gene family, now known to be within Rfp-Y is expressed. Zoorob et al., Eur. J. Immunol. 23:1139-45 (1993). Transcripts of an Mhc Class I gene within Rfp-Y were also found in many different tissues. See Afanassieff et al., Abstract presented at the Avian Immunology Research Group Meeting, Obergurgal, Austria, April 21-24, 1996; Afanassieff et al., in Kasahara, ed., The Major Histocompatibility Complex: Evolution, Structure, and Function, Springer-Verlag, New York, 2000, pp. 236-247. Nevertheless, the role of the Rfp-Y system in immune mediation of disease resistance and the extent to which genes of the Rfp-Y system are expressed in various cell types has heretofore remained unknown.
In accordance with the present invention, it has been discovered that the Rfp-Y system exerts an effect on Marek""s disease resistance. It has also been discovered that, in some allelic combinations, the influence of the Rfp-Y and B systems on Marek""s disease resistance may be additive or interactive. Accordingly, the invention provides a method for determining the haplotype of Rfp-Y and B systems of a domesticated fowl. This method involves obtaining a sample of nucleic acid such as genomic DNA from the fowl which contains a region having a sequence corresponding to an Rfp-Y, B-F, B-L or B-G region of the genome which is subject to allelic variation. B-F, B-L and B-G are all linked genes within the B system. The nucleic acid sample is amplified, and the resulting amplification products are denatured. These denatured amplification products are subjected to non-denaturing electrophoretic separation to produce an electrophoresis pattern that is characteristic of the Rfp-Y or B regions (B-F, B-L, B-G) of the domesticated fowl. In preferred embodiments, the method of the invention employs the polymerase chain reaction to produce amplification products of the Rfp-Y or B region of genomic DNA using primers that bound an allelic sequence in the region. Primers suitable for use in this inventive method bound sequences about 50 to about 500 nucleotides long, or preferably about 100 to about 300 nucleotides long and include those of SEQ ID NOS: 5-19. The invention provides primers of SEQ ID NOS:5-19 which may be used with these methods. The invention also provides methods of selecting and breeding disease-resistant domesticated fowl. Disease-resistant fowl are selected by determining the Rfp-Y or B haplotype of a population of domesticated fowl and correlating the haplotypes with resistance to a disease. Those birds possessing haplotypes correlated for disease resistance are selected for breeding and mated with birds of opposite gender to produce disease-resistant offspring.